Analysis of mRNA molecules derived from various genes is very important in order to elucidate biological phenomena. Discovery of an RNA-dependent DNA polymerase, so-called a reverse transcriptase, from a retrovirus has enabled a reverse transcription reaction in which a cDNA is synthesized using an RNA as a template. As a result, methods for analyzing mRNA molecules have made rapid progress. Then, the methods for analyzing mRNA molecules using a reverse transcriptase have now become indispensable experimental methods for studying genes. Furthermore, these methods, which have been applied to cloning techniques and PCR techniques, have become indispensable techniques not only for studying genes but also in wide variety of fields including biology, medicine and agriculture.
However, the conventional reverse transcription methods had many problems such as interruption of a cDNA synthesis reaction, inability to synthesize a long cDNA, low fidelity, and damage of a template RNA in the course of reaction due to long time required for the reaction.
It is considered that the interruption of the cDNA synthesis reaction is due to the secondary structure formed by the RNA as a template. Optimal reaction temperature for a reverse transcriptase from a retrovirus is low. RNAs form complicated secondary structures during the reaction at the temperature. Then, the cDNA synthesis reaction is interrupted at the sites of such secondary structures. A method in which a heat-resistant reverse transcriptase is used has been proposed in order to solve the above-mentioned problem. However, the reactivity of this method is not satisfactory. In addition, reverse transcriptases have not been known to exhibit a proofreading activity during a reverse transcription reaction, and a method for synthesizing a cDNA with high fidelity has not been known.
As described above, it was difficult to synthesize a cDNA with high efficiency and with high fidelity according to the conventional methods. Thus, a more efficient method for synthesizing a cDNA has been desired.